Search

Executive summary: Microbes are all pervasive in their distribution and influence on the functioning and well‐being of humans, life in general and the planet. Microbially‐based technologies contribute hugely to the supply of important goods and services we depend upon, such as the provision of food, medicines and clean water. They also offer mechanisms and strategies to mitigate and solve a wide range of problems and crises facing humanity at all levels, including those encapsulated in the sustainable development goals (SDGs) formulated by the United Nations. For example, microbial technologies can contribute in multiple ways to decarbonisation and hence confronting global warming, provide sanitation and clean water to the billions of people lacking them, improve soil fertility and hence food production and develop vaccines and other medicines to reduce and in some cases eliminate deadly infections. They are the foundation of biotechnology, an increasingly important and growing business sector and source of employment, and the centre of the bioeconomy, Green Deal, etc. But, because microbes are largely invisible, they are not familiar to most people, so opportunities they offer to effectively prevent and solve problems are often missed by decision‐makers, with the negative consequences this entrains. To correct this lack of vital knowledge, the International Microbiology Literacy Initiative–the IMiLI–is recruiting from the global microbiology community and making freely available, teaching resources for a curriculum in societally relevant microbiology that can be used at all levels of learning. Its goal is the development of a society that is literate in relevant microbiology and, as a consequence, able to take full advantage of the potential of microbes and minimise the consequences of their negative activities. In addition to teaching about microbes, almost every lesson discusses the influence they have on sustainability and the SDGs and their ability to solve pressing problems of societal inequalities. The curriculum thus teaches about sustainability, societal needs and global citizenship. The lessons also reveal the impacts microbes and their activities have on our daily lives at the personal, family, community, national and global levels and their relevance for decisions at all levels. And, because effective, evidence‐based decisions require not only relevant information but also critical and systems thinking, the resources also teach about these key generic aspects of deliberation. The IMiLI teaching resources are learner‐centric, not academic microbiology‐centric and deal with the microbiology of everyday issues. These span topics as diverse as owning and caring for a companion animal, the vast range of everyday foods that are produced via microbial processes, impressive geological formations created by microbes, childhood illnesses and how they are managed and how to reduce waste and pollution. They also leverage the exceptional excitement of exploration and discovery that typifies much progress in microbiology to capture the interest, inspire and motivate educators and learners alike. The IMiLI is establishing Regional Centres to translate the teaching resources into regional languages and adapt them to regional cultures, and to promote their use and assist educators employing them. Two of these are now operational. The Regional Centres constitute the interface between resource creators and educators–learners. As such, they will collect and analyse feedback from the end‐users and transmit this to the resource creators so that teaching materials can be improved and refined, and new resources added in response to demand: educators and learners will thereby be directly involved in evolution of the teaching resources. The interactions between educators–learners and resource creators mediated by the Regional Centres will establish dynamic and synergistic relationships–a global societally relevant microbiology education ecosystem–in which creators also become learners, teaching resources are optimised and all players/stakeholders are empowered and their motivation increased. The IMiLI concept thus embraces the principle of teaching societally relevant microbiology embedded in the wider context of societal, biosphere and planetary needs, inequalities, the range of crises that confront us and the need for improved decisioning, which should ultimately lead to better citizenship and a humanity that is more sustainable and resilient. The biosphere of planet Earth is a microbial world: a vast reactor of countless microbially driven chemical transformations and energy transfers that push and pull many planetary geochemical processes, including the cycling of the elements of life, mitigate or amplify climate change (e.g., Nature Reviews Microbiology, 2019, 17, 569) and impact the well‐being and activities of all organisms, including humans. Microbes are both our ancestors and creators of the planetary chemistry that allowed us to evolve (e.g., Life's engines: How microbes made earth habitable, 2023). To understand how the biosphere functions, how humans can influence its development and live more sustainably with the other organisms sharing it, we need to understand the microbes. In a recent editorial (Environmental Microbiology, 2019, 21, 1513), we advocated for improved microbiology literacy in society. Our concept of microbiology literacy is not based on knowledge of the academic subject of microbiology, with its multitude of component topics, plus the growing number of additional topics from other disciplines that become vitally important elements of current microbiology. Rather it is focused on microbial activities that impact us–individuals/communities/nations/the human world–and the biosphere and that are key to reaching informed decisions on a multitude of issues that regularly confront us, ranging from personal issues to crises of global importance. In other words, it is knowledge and understanding essential for adulthood and the transition to it, knowledge and understanding that must be acquired early in life in school. The 2019 Editorial marked the launch of the International Microbiology Literacy Initiative, the IMiLI. Here, we present: our concept of how microbiology literacy may be achieved and the rationale underpinning it;the type of teaching resources being created to realise the concept and the framing of microbial activities treated in these resources in the context of sustainability, societal needs and responsibilities and decision‐making; andthe key role of Regional Centres that will translate the teaching resources into local languages, adapt them according to local cultural needs, interface with regional educators and develop and serve as hubs of microbiology literacy education networks. The topics featuring in teaching resources are learner‐centric and have been selected for their inherent relevance, interest and ability to excite and engage. Importantly, the resources coherently integrate and emphasise the overarching issues of sustainability, stewardship and critical thinking and the pervasive interdependencies of processes. More broadly, the concept emphasises how the multifarious applications of microbial activities can be leveraged to promote human/animal, plant, environmental and planetary health, improve social equity, alleviate humanitarian deficits and causes of conflicts among peoples and increase understanding between peoples (Microbial Biotechnology, 2023, 16(6), 1091–1111). Importantly, although the primary target of the freely available (CC BY‐NC 4.0) IMiLI teaching resources is schoolchildren and their educators, they and the teaching philosophy are intended for all ages, abilities and cultural spectra of learners worldwide: in university education, lifelong learning, curiosity‐driven, web‐based knowledge acquisition and public outreach. The IMiLI teaching resources aim to promote development of a global microbiology education ecosystem that democratises microbiology knowledge. [ABSTRACT FROM AUTHOR]

DNA methylation serves a variety of functions across all life domains. In this study, we investigated archaeal methylomics within a tripartite xylanolytic halophilic consortium. This consortium includes Haloferax lucertense SVX82, Halorhabdus sp. SVX81, and an ectosymbiotic Candidatus Nanohalococcus occultus SVXNc, a nano‐sized archaeon from the DPANN superphylum. We utilized PacBio SMRT and Illumina cDNA sequencing to analyse samples from consortia of different compositions for methylomics and transcriptomics. Endogenous cTAG methylation, typical of Haloferax, was accompanied in this strain by methylation at four other motifs, including GDGcHC methylation, which is specific to the ectosymbiont. Our analysis of the distribution of methylated and unmethylated motifs suggests that autochthonous cTAG methylation may influence gene regulation. The frequency of GRAGAaG methylation increased in highly expressed genes, while CcTTG and GTCGaGG methylation could be linked to restriction‐modification (RM) activity. Generally, the RM activity might have been reduced during the evolution of this archaeon to balance the protection of cells from intruders, the reduction of DNA damage due to self‐restriction in stressful environments, and the benefits of DNA exchange under extreme conditions. Our methylomics, transcriptomics and complementary electron cryotomography (cryo‐ET) data suggest that the nanohaloarchaeon exports its methyltransferase to methylate the Haloferax genome, unveiling a new aspect of the interaction between the symbiont and its host. [ABSTRACT FROM AUTHOR]

Climate change, desertification, salinisation of soils and the changing hydrology of the Earth are creating or modifying microbial habitats at all scales including the oceans, saline groundwaters and brine lakes. In environments that are saline or hypersaline, the biodegradation of recalcitrant plant and animal polysaccharides can be inhibited by salt‐induced microbial stress and/or by limitation of the metabolic capabilities of halophilic microbes. We recently demonstrated that the chitinolytic haloarchaeon Halomicrobium can serve as the host for an ectosymbiont, nanohaloarchaeon 'Candidatus Nanohalobium constans'. Here, we consider whether nanohaloarchaea can benefit from the haloarchaea‐mediated degradation of xylan, a major hemicellulose component of wood. Using samples of natural evaporitic brines and anthropogenic solar salterns, we describe genome‐inferred trophic relations in two extremely halophilic xylan‐degrading three‐member consortia. We succeeded in genome assembly and closure for all members of both xylan‐degrading cultures and elucidated the respective food chains within these consortia. We provide evidence that ectosymbiontic nanohaloarchaea is an active ecophysiological component of extremely halophilic xylan‐degrading communities (although by proxy) in hypersaline environments. In each consortium, nanohaloarchaea occur as ectosymbionts of Haloferax, which in turn act as scavenger of oligosaccharides produced by xylan‐hydrolysing Halorhabdus. We further obtained and characterised the nanohaloarchaea–host associations using microscopy, multi‐omics and cultivation approaches. The current study also doubled culturable nanohaloarchaeal symbionts and demonstrated that these enigmatic nano‐sized archaea can be readily isolated in binary co‐cultures using an appropriate enrichment strategy. We discuss the implications of xylan degradation by halophiles in biotechnology and for the United Nation's Sustainable Development Goals. [ABSTRACT FROM AUTHOR]

Practical experiments drive important scientific discoveries in biology, but theory‐based research studies also contribute novel—sometimes paradigm‐changing—findings. Here, we appraise the roles of theory‐based approaches focusing on the experiment‐dominated wet‐biology research areas of microbial growth and survival, cell physiology, host–pathogen interactions, and competitive or symbiotic interactions. Additional examples relate to analyses of genome‐sequence data, climate change and planetary health, habitability, and astrobiology. We assess the importance of thought at each step of the research process; the roles of natural philosophy, and inconsistencies in logic and language, as drivers of scientific progress; the value of thought experiments; the use and limitations of artificial intelligence technologies, including their potential for interdisciplinary and transdisciplinary research; and other instances when theory is the most‐direct and most‐scientifically robust route to scientific novelty including the development of techniques for practical experimentation or fieldwork. We highlight the intrinsic need for human engagement in scientific innovation, an issue pertinent to the ongoing controversy over papers authored using/authored by artificial intelligence (such as the large language model/chatbot ChatGPT). Other issues discussed are the way in which aspects of language can bias thinking towards the spatial rather than the temporal (and how this biased thinking can lead to skewed scientific terminology); receptivity to research that is non‐mainstream; and the importance of theory‐based science in education and epistemology. Whereas we briefly highlight classic works (those by Oakes Ames, Francis H.C. Crick and James D. Watson, Charles R. Darwin, Albert Einstein, James E. Lovelock, Lynn Margulis, Gilbert Ryle, Erwin R.J.A. Schrödinger, Alan M. Turing, and others), the focus is on microbiology studies that are more‐recent, discussing these in the context of the scientific process and the types of scientific novelty that they represent. These include several studies carried out during the 2020 to 2022 lockdowns of the COVID‐19 pandemic when access to research laboratories was disallowed (or limited). We interviewed the authors of some of the featured microbiology‐related papers and—although we ourselves are involved in laboratory experiments and practical fieldwork—also drew from our own research experiences showing that such studies can not only produce new scientific findings but can also transcend barriers between disciplines, act counter to scientific reductionism, integrate biological data across different timescales and levels of complexity, and circumvent constraints imposed by practical techniques. In relation to urgent research needs, we believe that climate change and other global challenges may require approaches beyond the experiment. [ABSTRACT FROM AUTHOR]

Summary: Halorhodospira halophila, one of the most‐xerophilic halophiles, inhabits biophysically stressful and energetically expensive, salt‐saturated alkaline brines. Here, we report an additional stress factor that is biotic: a diminutive Candidate‐Phyla‐Radiation bacterium, that we named 'Ca. Absconditicoccus praedator' M39‐6, which predates H. halophila M39‐5, an obligately photosynthetic, anaerobic purple‐sulfur bacterium. We cultivated this association (isolated from the hypersaline alkaline Lake Hotontyn Nur, Mongolia) and characterized their biology. 'Ca. Absconditicoccus praedator' is the first stably cultivated species from the candidate class‐level lineage Gracilibacteria (order‐level lineage Absconditabacterales). Its closed‐and‐curated genome lacks genes for the glycolytic, pentose phosphate‐ and Entner–Doudoroff pathways which would generate energy/reducing equivalents and produce central carbon currencies. Therefore, 'Ca. Absconditicoccus praedator' is dependent on host‐derived building blocks for nucleic acid‐, protein‐, and peptidoglycan synthesis. It shares traits with (the uncultured) 'Ca. Vampirococcus lugosii', which is also of the Gracilibacteria lineage. These are obligate parasitic lifestyle, feeding on photosynthetic anoxygenic Gammaproteobacteria, and absorption of host cytoplasm. Commonalities in their genomic composition and structure suggest that the entire Absconditabacterales lineage consists of predatory species which act to cull the populations of their respective host bacteria. Cultivation of vampire : host associations can shed light on unresolved aspects of their metabolism and ecosystem dynamics at life‐limiting extremes. [ABSTRACT FROM AUTHOR]

Summary: Astrobiology is mistakenly regarded by some as a field confined to studies of life beyond Earth. Here, we consider life on Earth through an astrobiological lens. Whereas classical studies of microbiology historically focused on various anthropocentric sub‐fields (such as fermented foods or commensals and pathogens of crop plants, livestock and humans), addressing key biological questions via astrobiological approaches can further our understanding of all life on Earth. We highlight potential implications of this approach through the articles in this Environmental Microbiology special issue 'Ecophysiology of Extremophiles'. They report on the microbiology of places/processes including low‐temperature environments and chemically diverse saline‐ and hypersaline habitats; aspects of sulphur metabolism in hypersaline lakes, dysoxic marine waters, and thermal acidic springs; biology of extremophile viruses; the survival of terrestrial extremophiles on the surface of Mars; biological soils crusts and rock‐associated microbes of deserts; subsurface and deep biosphere, including a salticle formed within Triassic halite; and interactions of microbes with igneous and sedimentary rocks. These studies, some of which we highlight here, contribute to our understanding of the spatiotemporal reach of Earth'sfunctional biosphere, and the tenacity of terrestrial life. Their findings will help set the stage for future work focused on the constraints for life, and how organisms adapt and evolve to circumvent these constraints. [ABSTRACT FROM AUTHOR]

Summary: Archaea are environmentally ubiquitous on Earth, and their extremophilic and metabolically versatile phenotypes make them useful as model systems for astrobiology. Here, we reveal a new functional group of halo(natrono)archaea able to utilize alpha‐d‐glucans (amylopectin, amylose and glycogen), sugars, and glycerol as electron donors and carbon sources for sulfur respiration. They are facultative anaerobes enriched from hypersaline sediments with either amylopectin, glucose or glycerol as electron/carbon sources and elemental sulfur as the terminal electron acceptor. They include 10 strains of neutrophilic haloarchaea from circum pH‐neutral lakes and one natronoarchaeon from soda‐lake sediments. The neutrophilic isolates can grow by fermentation, although addition of S0 or dimethyl sulfoxide increased growth rate and biomass yield (with a concomitant decrease in H2). Natronoarchaeal isolate AArc‐S grew only by respiration, either anaerobically with S0 or thiosulfate as the terminal electron acceptor, or aerobically. Through genome analysis of five representative strains, we detected the full set of enzymes required for the observed catabolic and respiratory phenotypes. These findings provide evidence that sulfur‐respiring haloarchaea partake in biogeochemical sulfur cycling, linked to terminal anaerobic carbon mineralization in hypersaline anoxic habitats. We discuss the implications for life detection in analogue environments such as the polar subglacial brine‐lakes of Mars. [ABSTRACT FROM AUTHOR]

A rich microbiota in the deep ‐ sea oceans has evolved to live off petroleum from natural oil seeps. Understanding their ecology and biology could inform measures for sustainable bioremediation of artificial oil spills. [ABSTRACT FROM AUTHOR]

Marine prokaryotes have evolved a broad repertoire of defence systems to protect their genomes from lateral gene transfer including innate or acquired immune systems and infection-induced programmed cell suicide and dormancy. Here we report on the analysis of multiple defence systems present in the genome of the strain Cycloclasticus sp. 78-ME isolated from petroleum deposits of the tanker 'Amoco Milford Haven'. Cycloclasticus are ubiquitous bacteria globally important in polyaromatic hydrocarbons degradation in marine environments. Two 'defence islands' were identified in 78-ME genome: the first harbouring CRISPR-Cas with toxin-antitoxin system, while the second was composed by an array of genes for toxin-antitoxin and restriction-modification proteins. Among all identified spacers of CRISPR-Cas system only seven spacers match sequences of phages and plasmids. Furthermore, a conjugative plasmid p7ME01, which belongs to a new IncP-1h ancestral archetype without any accessory mobile elements was found in 78-ME. Our results provide the context to the co-occurrence of diverse defence mechanisms in the genome of Cycloclasticus sp. 78-ME, which protect the genome of this highly specialized PAH-degrader. This study contributes to the further understanding of complex networks established in petroleum-based microbial communities. [ABSTRACT FROM AUTHOR]

Dark ocean microbial communities are actively involved in chemoautotrophic and anaplerotic fixation of bicarbonate. Thus, aphotic pelagic realm of the ocean might represent a significant sink of CO2 and source of primary production. However, the estimated metabolic activities in the dark ocean are fraught with uncertainties. Typically, deep-sea samples are recovered to the sea surface for downstream processing on deck. Shifts in ambient settings, associated with such treatments, can likely change the metabolic activity and community structure of deep-sea adapted autochthonous microbial populations. To estimate influence of recovery and short-term handling of deep-sea samples, we monitored the succession of bathypelagic microbial community during its 3 days long on deck incubation. We demonstrated that at the end of exposition, the deep-sea archaeal population decreased threefold, whereas the bacterial fraction doubled in size. As revealed by phylogenetic analyses of amoA gene transcripts, dominance of the active ammonium-oxidizing bathypelagic T haumarchaeota groups shifted over time very fast. These findings demonstrated the simultaneous existence of various 'deep-sea ecotypes', differentially reacting to the sampling and downstream handling. Our study supports the hypothesis that metabolically active members of meso- and bathypelagic T haumarchaeota possess the habitat-specific distribution, metabolic complexity and genetic divergence at subpopulation level. [ABSTRACT FROM AUTHOR]

Within the complex of deep, hypersaline anoxic lakes (DHALs) of the Mediterranean Ridge, we identified a new, unexplored DHAL and named it 'Lake Kryos' after a nearby depression. This lake is filled with magnesium chloride (MgCl2)-rich, athalassohaline brine (salinity > 470 practical salinity units), presumably formed by the dissolution of Messinian bischofite. Compared with the DHAL Discovery, it contains elevated concentrations of kosmotropic sodium and sulfate ions, which are capable of reducing the net chaotropicily of MgCl2-rich solutions. The brine of Lake Kryos may therefore be biologically permissive at MgCl2 concentrations previously considered incompatible with life. We characterized the microbiology of the seawater-Kryos brine interface and managed to recover mRNA from the 2.27-3.03 M MgCl2 layer (equivalent to 0.747-0.631 water activity), thereby expanding the established chaotropicity window-for-life. The primary bacterial taxa present there were Kebrit Deep Bacteria 1 candidate division and DHAL-specific group of organisms, distantly related to Desulfohalobium. Two euryarchaeal candidate divisions, Mediterranean Sea Brine Lakes group 1 and halophilic cluster 1, accounted for > 85% of the rRNA-containing archaeal clones derived from the 2.27-3.03 M MgCl2 layer, but were minority community-members in the overlying interfacelayers. These findings shed light on the plausibility of life in highly chaotropic environments, geochemical windows for microbial extremophiles, and have implications for habitability elsewhere in the Solar System. [ABSTRACT FROM AUTHOR]

Since a key requirement of known life forms is available water (water activity; aw), recent searches for signatures of past life in terrestrial and extraterrestrial environments have targeted places known to have contained significant quantities of biologically available water. However, early life on Earth inhabited high-salt environments, suggesting an ability to withstand low water-activity. The lower limit of water activity that enables cell division appears to be ~ 0.605 which, until now, was only known to be exhibited by a single eukaryote, the sugar-tolerant, fungal xerophile Xeromyces bisporus. The first forms of life on Earth were, though, prokaryotic. Recent evidence now indicates that some halophilic Archaea and Bacteria have water-activity limits more or less equal to those of X. bisporus. We discuss water activity in relation to the limits of Earth's present-day biosphere; the possibility of microbial multiplication by utilizing water from thin, aqueous films or non-liquid sources; whether prokaryotes were the first organisms able to multiply close to the 0.605-aw limit; and whether extraterrestrial aqueous milieux of ≥ 0.605 aw can resemble fertile microbial habitats found on Earth. [ABSTRACT FROM AUTHOR]

Ammonium-oxidizing chemoautotrophic members of T haumarchaea are proposed to be the key players in the assimilation of bicarbonate in the dark ( ABD). However, this process may also involve heterotrophic metabolic pathways, such as fixation of carbon dioxide ( CO2) via various anaplerotic reactions. We collected samples from the depth of 4900 m at the Matapan- Vavilov Deep ( MVD) station ( Hellenic Trench, Eastern Mediterranean) and used the multiphasic approach to study the ABD mediators in this deep-sea ecosystem. At this depth, our analysis indicated the occurrence of actively CO2-fixing heterotrophic microbial assemblages dominated by G ammaproteobacteria with virtually no T haumarchaea present. [ 14C]-bicarbonate incorporation experiments combined with shotgun [ 14C]-proteomic analysis identified a series of proteins of gammaproteobacterial origin. More than quarter of them were closely related with A lteromonas macleodii 'deep ecotype' AltDE, the predominant organism in the microbial community of MVD. The present study demonstrated that in the aphotic/hadal zone of the Mediterranean Sea, the assimilation of bicarbonate is associated with both chemolithoauto- and heterotrophic ABD. In some deep-sea areas, the latter may predominantly contribute to the de novo synthesis of organic carbon which points at the important and yet underestimated role heterotrophic bacterial populations can play the in global carbon cycle/sink in the ocean interior. [ABSTRACT FROM AUTHOR]

Euryarchaea from the genus H alorhabdus have been found in hypersaline habitats worldwide, yet are represented by only two isolates: H alorhabdus utahensis AX-2T from the shallow Great Salt Lake of Utah, and H alorhabdus tiamatea SARL4 BT from the Shaban deep-sea hypersaline anoxic lake ( DHAL) in the Red Sea. We sequenced the H . tiamatea genome to elucidate its niche adaptations. Among sequenced archaea, H . tiamatea features the highest number of glycoside hydrolases, the majority of which were expressed in proteome experiments. Annotations and glycosidase activity measurements suggested an adaptation towards recalcitrant algal and plant-derived hemicelluloses. Glycosidase activities were higher at 2% than at 0% or 5% oxygen, supporting a preference for low-oxygen conditions. Likewise, proteomics indicated quinone-mediated electron transport at 2% oxygen, but a notable stress response at 5% oxygen. H alorhabdus tiamatea furthermore encodes proteins characteristic for thermophiles and light-dependent enzymes (e.g. bacteriorhodopsin), suggesting that H . tiamatea evolution was mostly not governed by a cold, dark, anoxic deep-sea habitat. Using enrichment and metagenomics, we could demonstrate presence of similar glycoside hydrolase-rich H alorhabdus members in the Mediterranean DHAL Medee, which supports that H alorhabdus species can occupy a distinct niche as polysaccharide degraders in hypersaline environments. [ABSTRACT FROM AUTHOR]

We used a combination of molecular and microbiological approaches to determine the activity, abundance and diversity of archaeal populations inhabiting meromictic saline Lake Faro ( Messina, Italy). Analysis of archaeal 16 S rRNA, amoA, accA and hbd genes and transcripts revealed that sub- and anoxic layers of Lake Faro are primarily inhabited by the organisms related to the clusters of Marine Group I.1a of Thaumarchaeota frequently recovered from oxygen-depleted marine ecosystems. These organisms dominated the metabolically active archaea down to the bottom of the lake, indicating their adaptation to recurrent changes in the levels of water column hypoxia. The upper microaerobic layer of Lake Faro redoxcline has the maximal rates of dark primary production much lower than those of other previously studied pelagic redoxclines, but comparable to the values of meso- and bathypelagic areas of Mediterranean Sea. Application of bacterial inhibitors, especially azide, significantly declined the CO2 fixation rates in the low interface and monimolimnion, whereas archaea-specific inhibitor had effect only in upper part of the redoxcline. Based on these findings, we hypothesize that dark bicarbonate fixation in suboxic zone of Lake Faro results mainly from archaeal activity which is affected by the predicted lack in oxygen in lower layers. [ABSTRACT FROM AUTHOR]

The marine pelagic zone situated > 200 m below the sea level (bls) is the largest marine subsystem, comprising more than two-thirds of the oceanic volume. At the same time, it is one of the least explored ecosystems on Earth. Few large-scale environmental genomics studies have been undertaken to examine the phylogenetic diversity and functional gene repertoire of planktonic microbes present in mesopelagic and bathypelagic environments. Here, we present the description of the deep-sea microbial community thriving at > 4900 m depth in Matapan-Vavilov Deep (MVD). This canyon is the deepest site of Mediterranean Sea, with a deepest point located at approximately 5270 m, 56 km SW of city Pylos (Greece) in the Ionian Sea (36°34.00N, 21°07.44E). Comparative analysis of whole-metagenomic data revealed that unlike other deep-sea metagenomes, the prokaryotic diversity in MVD was extremely poor. The decline in the dark primary production rates, measured at 4908 m depth, was coincident with overwhelming dominance of copiotrophic Alteromonas macleodii'deep-ecotype' AltDE at the expense of other prokaryotes including those potentially involved in both autotrophic and anaplerotic CO2 fixation. We also demonstrate the occurrence in deep-sea metagenomes of several clustered regularly interspaced short palindromic repeats systems. [ABSTRACT FROM AUTHOR]

Summary So far only little is known about the microbial ecology of Mediterranean deep-sea hypersaline anoxic lakes (DHALs). These brine lakes were formed by evaporite dissolution/brine seeps and are important model environments to provide insights into possible metabolisms and distributions of microorganisms on the early Earth. Our study on the Lake Thetis, a new thalassohaline DHAL located South-East of the Medriff Corridor, has revealed microbial communities of contrasting compositions with a high number of novel prokaryotic candidate divisions. The major finding of our present work is co-occurrence of at least three autotrophic carbon dioxide fixation pathways in the brine-seawater interface that are likely fuelled by an active ramified sulphur cycle. Genes for the reductive acetyl-CoA and reductive TCA pathways were also found in the brine suggesting that these pathways are operational even at extremely elevated salinities and that autotrophy is more important in hypersaline environments than previously assumed. Surprisingly, genes coding for RuBisCo were found in the highly reduced brine. Three types of sulphide oxidation pathways were found in the interface. The first involves a multienzyme Sox complex catalysing the complete oxidation of reduced sulphur compounds to sulphate, the second type recruits SQR sulphide:quinone reductase for oxidation of sulphide to elemental sulphur, which, in the presence of sulphide, could further be reduced by polysulphide reductases in the third pathway. The presence of the latter two allows a maximal energy yield from the oxidation of sulphide and at the same time prevents the acidification and the accumulation of S0 deposits. Amino acid composition analysis of deduced proteins revealed a significant overrepresentation of acidic residues in the brine compared with the interface. This trait is typical for halophilic organisms as an adaptation to the brine's extreme hypersalinity. This work presents the first metagenomic survey of the microbial communities of the recently discovered Lake Thetis whose brine constitutes one of saltiest water bodies ever reported. [ABSTRACT FROM AUTHOR]

Summary In September 2008, an expedition of the RV Urania was devoted to exploration of the genomic richness of deep hypersaline anoxic lakes (DHALs) located in the Western part of the Mediterranean Ridge. Approximately 40 nautical miles SE from Urania Lake, the presence of anoxic hypersaline lake, which we named Thetis, was confirmed by swath bathymetry profiling and through immediate sampling casts. The brine surface of the Thetis Lake is located at a depth of 3258 m with a thickness of ∼157 m. Brine composition was found to be thalassohaline, saturated by NaCl with a total salinity of 348‰, which is one of highest value reported for DHALs. Similarly to other Mediterranean DHALs, seawater-brine interface of Thetis represents a steep pycno- and chemocline with gradients of salinity, electron donors and acceptors and posseses a remarkable stratification of prokaryotic communities, observed to be more metabolically active in the upper interface where redox gradient was sharper. [14C]-bicarbonate fixation analysis revealed that microbial communities are sustained by sulfur-oxidizing chemolithoautotrophic primary producers that thrive within upper interface. Besides microaerophilic autotrophy, heterotrophic sulfate reduction, methanogenesis and anaerobic methane oxidation are likely the predominant processes driving the ecosystem of Thetis Lake. [ABSTRACT FROM AUTHOR]

Marine Crenarchaeota, ubiquitous and abundant organisms in the oceans worldwide, remain metabolically uncharacterized, largely due to their low cultivability. Identification of candidate genes for bicarbonate fixation pathway in the Cenarchaeum symbiosum A was an initial step in understanding the physiology and ecology of marine Crenarchaeota. Recent cultivation and genome sequencing of obligate chemoautotrophic Nitrosopumilus maritimus SCM1 were a major breakthrough towards understanding of their functioning and provide a valuable model for experimental validation of genomic data. Here we present the identification of multiple key components of 3-hydroxipropionate/4-hydroxybutyrate cycle, the fifth pathway in carbon fixation, found in data sets of environmental sequences representing uncultivated superficial and bathypelagic Crenarchaeota from Sargasso sea (GOS data set) and KM3 (Mediterranean Sea) and ALOHA (Atlantic ocean) stations. These organisms are likely to use acetyl-CoA/propionyl-CoA carboxylase(s) as CO2-fixing enzyme(s) to form succinyl-CoA, from which one molecule of acetyl-CoA is regenerated via 4-hydroxybutyrate cleavage and another acetyl-CoA to be the pathway product. The genetic distinctiveness and matching sympatric abundance imply that marine crenarchaeal genotypes from the three different geographic sites share similar ecophysiological properties, and therefore may represent fundamental units of marine ecosystem functioning. To couple results of sequence comparison with the dark ocean primary production, dissolved inorganic carbon fixation rates were measured at KM3 Station (3000 m depth, Eastern Mediterranean Sea), i.e. at the same site and depth used for metagenomic library construction. [ABSTRACT FROM AUTHOR]

A new piezotolerant alkane-degrading bacterium ( Marinobacter hydrocarbonoclasticus strain #5) was isolated from deep (3475 m) Mediterranean seawater and grown at atmospheric pressure (0.1 MPa) and at 35 MPa with hexadecane as sole source of carbon and energy. Modification of the hydrostatic pressure influenced neither the growth rate nor the amount of degraded hexadecane (≈ 90%) during 13 days of incubation. However, the lipid composition of the cells sharply differed under both pressure conditions. At 0.1 MPa, M. hydrocarbonoclasticus #5 biosynthesized large amounts (≈ 62% of the total cellular lipids) of hexadecane-derived wax esters (WEs), which accumulated in the cells under the form of individual lipid bodies. Intracellular WEs were also synthesized at 35 MPa, but their proportion was half that at 0.1 MPa. This lower WE content at high pressure was balanced by an increase in the total cellular phospholipid content. The chemical composition of WEs formed under both pressure conditions also strongly differed. Saturated WEs were preferentially formed at 0.1 MPa whereas diunsaturated WEs dominated at 35 MPa. This increase of the unsaturation ratio of WEs resembled the one classically observed for bacterial membrane lipid homeostasis. Remarkably, the unsaturation ratio of membrane fatty acids of M. hydrocarbonoclasticus grown at 35 MPa was only slightly higher than at 0.1 MPa. Overall, the results suggest that intracellular WEs and phospholipids play complementary roles in the physiological adaptation of strain #5 to different hydrostatic pressures. [ABSTRACT FROM AUTHOR]

Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [( kcat/ Km)]CE/[( kcat/ Km)]FAE factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys281, Asp282, Asn316 and Lys317) situated close to the catalytic core (Ser143/Asp273/His305) and a deletion of a 34-AA–long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in kcat/ Km values) and enzymes with inverted specificity (( kcat/ Km)CE/( kcat/ Km)FAE ratio of ∼0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to −5.6 J mol−1), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to −13.7 J mol−1) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of ‘hot spot’ mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction. [ABSTRACT FROM AUTHOR]

The frontiers of eukaryote life in nature are still unidentified. In this study, we analysed protistan communities in the hypersaline (up to 365 g l−1 NaCl) anoxic L'Atalante deep-sea basin located in the eastern Mediterranean Sea. Targeting 18S ribosomal RNA retrieved from the basin's lower halocline (3501 m depth) we detected 279 protistan sequences that grouped into 42 unique phylotypes (99% sequence similarity). Statistical analyses revealed that these phylotypes account only for a proportion of the protists inhabiting this harsh environment with as much as 50% missed by this survey. Most phylotypes were affiliated with ciliates (45%), dinoflagellates (21%), choanoflagelates (10%) and uncultured marine alveolates (6%). Sequences from other taxonomic groups like stramenopiles, Polycystinea, Acantharea and Euglenozoa, all of which are typically found in non-hypersaline deep-sea systems, are either missing or very rare in our cDNA clone library. Although many DHAB sequences fell within previously identified environmental clades, a large number branched relatively deeply. Phylotype richness, community membership and community structure differ significantly from a deep seawater reference community (3499 m depth). Also, the protistan community in the L'Atalante basin is distinctively different from any previously described hypersaline community. In conclusion, we hypothesize that extreme environments may exert a high selection pressure possibly resulting in the evolution of an exceptional and distinctive assemblage of protists. The deep hypersaline anoxic basins in the Mediterranean Sea provide an ideal platform to test for this hypothesis and are promising targets for the discovery of undescribed protists with unknown physiological capabilities. [ABSTRACT FROM AUTHOR]

There is little information on how different strategies for the bioremediation of marine oil spills influence the key indigenous hydrocarbon-degrading bacteria (hydrocarbonoclastic bacteria, HCB), and hence their remediation efficacy. Therefore, we have used quantitative polymerase chain reaction to analyse changes in concentrations of HCB in response to intervention strategies applied to experimental microcosms. Biostimulation with nutrients (N and P) produced no measurable increase in either biodegradation or concentration of HCB within the first 5 days, but after 15 days there was a significant increase (29%; P < 0.05) in degradation of n-alkanes, and an increase of one order of magnitude in concentration of Thalassolituus (to 107 cells ml−1). Rhamnolipid bioemulsifier additions alone had little effect on biodegradation, but, in combination with nutrient additions, provoked a significant increase: 59% ( P < 0.05) more n-alkane degradation by 5 days than was achieved with nutrient additions alone. The very low Alcanivorax cell concentrations in the microcosms were hardly influenced by addition of nutrients or bioemulsifier, but strongly increased after their combined addition, reflecting the synergistic action of the two types of biostimulatory agents. Bioaugmentation with Thalassolituus positively influenced hydrocarbon degradation only during the initial 5 days and only of the n-alkane fraction. Bioaugmentation with Alcanivorax was clearly much more effective, resulting in 73% greater degradation of n-alkanes, 59% of branched alkanes, and 28% of polynuclear aromatic hydrocarbons, in the first 5 days than that obtained through nutrient addition alone ( P < 0.01). Enhanced degradation due to augmentation with Alcanivorax continued throughout the 30-day period of the experiment. In addition to providing insight into the factors limiting oil biodegradation over time, and the competition and synergism between HCB, these results add weight to the use of bioaugmentation in oil pollution mitigation strategies. [ABSTRACT FROM AUTHOR]

The biosphere of planet Earth is delineated by physico-chemical conditions that are too harsh for, or inconsistent with, life processes and maintenance of the structure and function of biomolecules. To define the window of life on Earth (and perhaps gain insights into the limits that life could tolerate elsewhere), and hence understand some of the most unusual biological activities that operate at such extremes, it is necessary to understand the causes and cellular basis of systems failure beyond these windows. Because water plays such a central role in biomolecules and bioprocesses, its availability, properties and behaviour are among the key life-limiting parameters. Saline waters dominate the Earth, with the oceans holding 96.5% of the planet's water. Saline groundwater, inland seas or saltwater lakes hold another 1%, a quantity that exceeds the world's available freshwater. About one quarter of Earth's land mass is underlain by salt, often more than 100 m thick. Evaporite deposits contain hypersaline waters within and between their salt crystals, and even contain large subterranean salt lakes, and therefore represent significant microbial habitats. [ABSTRACT FROM AUTHOR]

An ecological study on distribution of Antarctic bacterial communities was determined by 16S-based phylogenetic analyses of clone libraries derived from RNA and DNA extracted from two different marine areas and compared between each other. Superficial seawater samples were collected from four stations in Ross Sea, three of them located in Rod Bay and one in Evans Cove; for each station two clone libraries (16S rDNA and 16S rRNA) were prepared and evident divergences between DNA and RNA libraries of each site were obtained. Of all phylotypes 93.6% were found in RNA libraries; in contrast, only 31 phylotypes (70.5%) were retrieved from total microbial community (DNA libraries). DNA and RNA sequences related to γ- Proteobacteria and Bacteroidetes groups, typical for Antarctic sea-ice bacterial communities, were detected in analysed sites. 16S rDNA and rRNA libraries derived from the two different areas were enriched by picophytoplanktonic 16S sequences of plastid and mitochondrion origins, reflecting that the algal blooms occurred during sampling (Antarctic summer 2003). The finding in Rod Bay libraries of high percentage of DNA clones apparently affiliated with β- Proteobacteria typical for activated sludges and well water could be explained by the presence of a sewage depuration system at this site. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA gene sequencing is preferred approach to have a more reliable vision on the composition of microbial communities. [ABSTRACT FROM AUTHOR]

A metagenome expression library of bulk DNA extracted from the rumen content of a dairy cow was established in a phage lambda vector and activity-based screening employed to explore the functional diversity of the microbial flora. Twenty-two clones specifying distinct hydrolytic activities (12 esterases, nine endo-β-1,4-glucanases and one cyclodextrinase) were identified in the library and characterized. Sequence analysis of the retrieved enzymes revealed that eight (36%) were entirely new and formed deep-branched phylogenetic lineages with no close relatives among known ester- and glycosyl-hydrolases. Bioinformatic analyses of the hydrolase gene sequences, and the sequences and contexts of neighbouring genes, suggested tentative phylogenetic assignments of the rumen organisms producing the retrieved enzymes. The phylogenetic novelty of the hydrolases suggests that some of them may have potential for new applications in biocatalysis. [ABSTRACT FROM AUTHOR]

Hydrocarbon-contaminated superficial sediments collected from the Harbor of Milazzo (Tirrenean Sea, northern Sicily), a zone strongly affected by anthropogenic activities, were examined for in situ biodegradative capacities. A culture-independent molecular phylogenetic approach was used to study the influence of hydrocarbon and nutrient addition on the activity and diversity of the indigenous microbiota during a microcosm evaluation. The autochthonous microbial community in non-polluted sediments was represented by eubacterial phylotypes grouped within Proteobacteria, CFB and Firmicutes. The archaeal domain was represented by members of Marine Group I of Crenarchaeota. The majority of recovered sequences was affiliated with heterotrophic genera Clostridium and Vibrio, typical members of eutrophic coastal environments. Amendments of hydrocarbons and mineral nutrients to microcosms dramatically changed the initial diversity of the microbial community. Only bacterial phylotypes affiliated with Proteobacteria and CFB division were detected. The decrease in diversity observed in several microcosms could be explained by the strong selection for microorganisms belonging to group of marine hydrocarbonoclastic γ- Proteobacteria, namely Alcanivorax, Cycloclasticus, Marinobacter, Marinobacterium/Neptunomonas and Thalassolituus. This study demonstrated that nutrient amendment to hydrocarbon-contaminated superficial sediments enhanced the indigenous microbial biodegradation activity and that highly specialized marine hydrocarbonoclastic bacteria, representing a minor fraction in the natural microbial community, play an important role in the biodegradation of petroleum hydrocarbons accidentally entering the coastal environment. [ABSTRACT FROM AUTHOR]

Summary In San Biagio of Belpasso, approximately 20 km south of Mt. Etna, in the area of contact between volcanic and sedimentary formations, a number of small (3– 60 cm in diameter) active mud eruptions discharge CO2 -rich gases, mud and NaCl brines. They can be described as mini-volcanoes owing to their typical conic shapes and continuously bubbling peak craters. Samples were collected from the active peak craters at a depth of 20 cm and DNA was immediately extracted and amplified with universal 16S rRNA gene-specific primers, followed by cloning procedure. A total of 140 bacterial clones obtained were screened and clustered by restriction fragment length polymorphism (RFLP) analysis. The pool of 16S rRNA sequences representing each RFLP cluster was subjected to phylogenetic analysis. All of the 33 sequences analysed were affiliated with the kingdom of Eubacteria ; 28 sequences (77% of all clones) affiliated with the Proteobacteria , two sequences (19% of all clones) were affiliated with Actinobacteria and three sequences (4% of all clones) were affiliated with the Flexibacter-Cytophaga-Bacteroides division. The data obtained suggest that the microorganisms phylogenetically affiliated to autotrophic methane oxidizers and heterotrophic hydrocarbon degraders belonging to the gamma-subclass of Proteobacteria are major constituents of the microbial communities of the saline volcanic muds. Overall, the composition of the microbial community of the San Biagio mud volcano resembles the compositions of marine microbial communities, which might indicate that wind-blown seawater vapour acted as an inoculum for microbial community described in present work. [ABSTRACT FROM AUTHOR]

Here, we propose an advanced method for recently developed fingerprinting strategies to analyse microbial populations by direct detection of 16S rRNA sequences occurring in natural habitats. The differential display (DD) technique, which is widely used to analyse for eukaryotic gene expression, was optimized to assess bacterial rRNA diversity in environmental samples. Double-stranded cDNAs of rRNAs were synthesized without a forward primer digested with endonuclease and ligated with a double-stranded adapter. The fragments obtained were then amplified using an adapter-specific extended primer and a 16S rDNA universal reverse primer pair displayed by electrophoresis on a polyacrylamide gel. We validated this approach by characterization of a microbial community colonizing a geothermal (48°C) vent system Iocated close to the eruption zone of the south-east crater of the Mount Etna volcano, Sicily. Analysis of the patterns of abundant 16S rRNA revealed a considerable diversity of metabolically active bacteria phylogenetically clustering within the Crenarchaeota, Cyanobacteria, Firmicutes, Planctomycetales and Thermus divisions. Two sequence phylotypes were affiliated with uncultivated representatives of the recently described candidate division OP10 from a Yellowstone hot spring. [ABSTRACT FROM AUTHOR]

High salt concentrations, absence of light, anoxia, and high hydrostatic pressure make deep hypersaline anoxic basins (DHABs) in the Eastern Mediterranean Sea one of the most polyextreme habitats on Earth. Taking advantage of the unique chemical characteristics of these basins, we tested the effect of environmental selection and geographic distance on the structure of protistan communities. Terminal restriction fragment length polymorphism (T-RFLP) analyses were performed on water samples from the brines and seawater/brine interfaces of five basins: Discovery, Urania, Thetis, Tyro, and Medee. Using statistical analyses, we calculated the partitioning of diversity among the ten individual terminal restriction fragment (T-RF) profiles, based on peak abundance and peak incidence. While a significant distance effect on spatial protistan patterns was not detected, hydrochemical gradients emerged as strong dispersal barriers that likely lead to environmental selection in the DHAB protistan plankton communities. We identified sodium, magnesium, sulfate, and oxygen playing in concerto as dominant environmental drivers for the structuring of protistan plankton communities in the Eastern Mediterranean DHABs., (© 2012 The Authors. Published by Blackwell Publishing Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)